CN111978385A

CN111978385A - Extraction method of edestin, product and application thereof

Info

Publication number: CN111978385A
Application number: CN201910844488.9A
Authority: CN
Inventors: 赵兵; 徐鹏伟; 赵庆生; 袁晓凡; 常坦然; 于朝晖
Original assignee: Yunnan Hanmeng Pharmaceutical Co ltd
Current assignee: Yunnan Hanmeng Pharmaceutical Co ltd
Priority date: 2019-09-06
Filing date: 2019-09-06
Publication date: 2020-11-24
Anticipated expiration: 2039-09-06
Also published as: WO2021042701A1; CN111978385B

Abstract

The invention relates to a method for extracting edestin, a product and application thereof. The extraction method comprises the steps of mixing the dehydrated and deoiled fructus cannabis with a salt solution, extracting, filtering and cooling to obtain the fructus cannabis globulin. The extraction method provided by the invention is simple and easy to operate, the extracted globulin has high yield and high purity, contains all essential amino acids of a human body, has particularly high arginine content, can be applied to food, health-care products or medicines, and has the effects of resisting fatigue, improving immune health, preventing cardiovascular diseases and the like.

Description

Extraction method of edestin, product and application thereof

Technical Field

The invention belongs to the technical field of protein extraction processes, and particularly relates to a method for extracting edestin, a product and application thereof.

Background

The hemp (hemp ) belongs to industrial hemp, and has a long planting history in China. Fructus Cannabis has no drug utilization value, and its seed fructus Cannabis has high edible value, and can be used as medicine. Fructus cannabis has homology of medicine and food, and the medicinal efficacy of fructus cannabis is from Shen nong's herbal Jing, and has the functions of moistening dryness, moistening intestines, treating stranguria and activating blood circulation. In recent years, fructus cannabis is widely used as a health preserving food material, a clinical medicine material and the like in China.

The fructus cannabis contains abundant oil (30-50%), the content of unsaturated fatty acid in the oil is higher than 85%, the ratio of omega-6 to omega-3 of linoleic acid and linolenic acid is between 2.29-4.68, and the fructus cannabis is beneficial to human health; lipid substances are mainly contained in human brain tissue, wherein omega-3 unsaturated fatty acids account for about 20% of the total amount, and omega-3 unsaturated fatty acids account for about 40% of the total amount of the lipid substances in human retina; fructus cannabis and contains gamma-linolenic acid (GLA) and stearidonic acid (SDA) which are rare in terrestrial plants, and can be converted into PG1, PG2 and PG3, and the three series of prostaglandins have important functions on human bodies.

Fructus Cannabis also rich in globulin (20% -30%), and fructus Cannabis globulin has molecular weight of 300000Da, and is composed of 6 subunits, each of which is composed of a basic group and an acidic group connected via disulfide bond. The amino acids composing the edestin are more than 20, 8 of which are essential amino acids for human body. Edestin is a very good globulin, similar to egg globulin. The application of fructus cannabis is reported.

CN100998413A discloses a new application of edestin in preparing functional food for assisting in reducing blood lipid. The hemp seed globulin is made into different forms of food by adopting a conventional process, and after the hemp seed globulin is directly eaten, the level of total cholesterol, triglyceride and Low Density Lipoprotein (LDL) in the blood of a human body can be stabilized, and simultaneously, the level of High Density Lipoprotein (HDL) is obviously increased, so that the hemp seed globulin plays a role in assisting in reducing blood fat. The hemp seed globulin product is safe and non-toxic, does not contain forbidden stimulant components such as stimulant, anesthetic, beta-blocker, diuretic, hormone medicine and the like, and has good application prospect.

CN100404069C discloses an application of edestin in preparing functional food. The product has the function of obviously improving the hemoglobin content and the number of red blood cells, can be used for producing anti-hypoxia functional food, and has good application prospect particularly in the aspect of preparing health-care functional food and sports nutritional functional food for improving hypoxia tolerance. The invention can be further processed into various edible food forms according to the conventional process, such as oral agents, tablets, powders, capsules, microcapsules, soft capsules, food additives, nutrition enhancers, baked products, beverages, milk substitutes, ice creams and the like.

In the prior art, functional reports on the edestin are numerous, but research on the extraction process is few, so that the development of the edestin extraction method which is simple and easy to operate, high in yield of the edestin, high in purity and good in functionality is significant.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an extraction method of edestin, and a product and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

In one aspect, the invention provides a method for extracting edestin, which comprises the steps of dehydrating and deoiling fructus cannabis, mixing with a salt solution, extracting, filtering and cooling to obtain the edestin.

The extraction method provided by the invention is simple and easy to operate, the extracted globulin has high yield and high purity, contains all essential amino acids of a human body, has particularly high arginine content, and has the effects of resisting fatigue, improving immune health, preventing cardiovascular diseases and the like. Because the solubility of the edestin in water is poor, the edestin is extracted in a salt solution with a certain concentration, so that the solubility can be ensured, and the yield and the purity can be ensured. The reason why the fructus cannabis is dehydrated and deoiled is that the fructus cannabis contains a large amount of grease, which reduces the extraction efficiency of protein and increases the separation cost in the later stage of protein separation, and the dehydration process is to avoid the loss of protein.

Preferably, the extraction method comprises the following steps:

(1) dehydrating and deoiling fructus cannabis;

(2) mixing the treated fructus cannabis with a salt solution for extraction to obtain an extracting solution;

(3) filtering the extracting solution obtained in the step (2), and then centrifuging to obtain a supernatant;

(4) And (4) standing and cooling the supernatant obtained in the step (3), and separating out protein to obtain the edestin.

Preferably, the deoiling manner in step (1) includes any one or a combination of at least two of a mechanical method, a solvent extraction method and a supercritical extraction method, for example, a combination of a mechanical method and a solvent extraction method, a combination of a solvent extraction method and a supercritical extraction method, a combination of a mechanical method and a supercritical extraction method, and the like, and any other feasible combination manner is not repeated herein.

Preferably, the mechanical process comprises a mechanical cold pressing process.

Preferably, the solvent extraction method comprises n-hexane and/or petroleum ether.

Preferably, the dehydrated and deoiled fructus cannabis processed in the step (1) has a water content of 5-10%, such as 5%, 6%, 7%, 8%, 9%, or 10%, etc., and an oil content of 3-5%, such as 3%, 3.5%, 4%, 4.5%, or 5%, etc.

After dehydration and deoiling treatment, the water content of the fructus cannabis is required to be within the range of 5-10%, and the oil content is within the range of 3-5%, so that the yield and the purity of the globulin can reach higher levels.

Preferably, the solvent of the salt solution in step (2) is water, and the solute includes any one or a combination of at least two of sodium chloride, ammonium sulfate, magnesium chloride, calcium chloride, and sodium sulfate, for example, a combination of sodium chloride and ammonium sulfate, a combination of magnesium chloride and calcium chloride, a combination of calcium chloride and sodium sulfate, and the like, and any other feasible combination modes are not repeated herein.

Preferably, the solvent of the salt solution in step (2) is water, and the solute comprises sodium chloride and ammonium sulfate.

The preferred solutes are sodium chloride and ammonium sulfate because the mixed salt solution system of sodium chloride and ammonium sulfate allows the extraction yield and purity of globulin to be optimized relative to other salt solution systems. Wherein the purity of sodium chloride and ammonium sulfate is higher in the following range of the mass concentration.

Preferably, the sodium chloride is present in the salt solution at a mass concentration of 3-6%, such as 3%, 4%, 5%, or 6%, etc., and the ammonium sulfate is present in the salt solution at a mass concentration of 1-4%, such as 1%, 2%, 3%, or 4%, etc.

Preferably, the salt solution in step (2) further contains hyaluronic acid and/or trehalose.

The hyaluronic acid and/or trehalose substances are added into the salt solution system to obviously improve the purity of the globulin, and the hyaluronic acid and/or trehalose possibly plays a role in stabilizing the protein. And the effects are more preferable within the following numerical ranges.

Preferably, the mass fraction of the hyaluronic acid in the salt solution is 0.1-1%, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.

Preferably, the trehalose is present in the salt solution in a mass fraction of 0.1-1%, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.

Preferably, the mass-to-volume ratio of the fructus cannabis to the saline solution after the treatment in step (2) is 1 (5-15) g/mL, such as 1:5g/mL, 1:6g/mL, 1:7g/mL, 1:8g/mL, 1:9g/mL, 1:10g/mL, 1:11g/mL, 1:13g/mL or 1:15g/mL, etc.

During extraction, the mass-volume ratio of the fructus cannabis to the salt solution, namely the feed-liquid ratio, is also a key factor influencing the protein extraction rate or purity, and the mass-volume ratio of the fructus cannabis to the salt solution is specifically selected to be 1 (5-15) g/mL, because the extraction rate can be remarkably improved under the condition that the extracted protein purity is kept similar in the range.

Preferably, the extraction in the step (2) comprises a two-stage extraction process, wherein the first-stage extraction mode is heating extraction, and the second-stage extraction mode is ultrasonic extraction.

The invention creatively discovers that compared with a single extraction mode, the two-stage extraction mode (firstly heating extraction and then ultrasonic extraction) is adopted, so that the extracted globulin is better in both yield and purity.

Preferably, the temperature of the heating extraction is 60-80 deg.C, such as 60 deg.C, 62 deg.C, 65 deg.C, 68 deg.C, 70 deg.C, 72 deg.C, 75 deg.C, 78 deg.C or 80 deg.C.

Preferably, the heating extraction time is 20-40min, such as 20min, 22min, 25min, 28min, 30min, 32min, 35min or 40 min.

Preferably, the temperature of the ultrasonic extraction is 30-40 ℃, such as 30 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 38 ℃ or 40 ℃ and the like.

Preferably, the time of the ultrasonic extraction is 60-180min, such as 60min, 70min, 80min, 90min, 100min, 110min, 120min, 140min, 150min, 160min or 180min, etc.

Preferably, the power of the ultrasound is 300-1000W/L, such as 300W/L, 400W/L, 500W/L, 600W/L, 700W/L, 800W/L, 900W/L or 1000W/L, etc., preferably 800W/L.

The temperature and time of the heating and ultrasonic processes are required to be within the above numerical range to achieve better yield and purity of the globulin.

Preferably, the cooling in step (4) means cooling to 2-6 ℃, such as 2 ℃, 3 ℃, 4 ℃, 5 ℃ or 6 ℃, etc.

The cooling temperature is one of the key factors affecting protein precipitation, i.e. protein yield.

Preferably, the protein of step (4) is precipitated and then centrifuged and washed.

Preferably, the washing comprises washing with an aqueous solution of methylcellulose followed by an aqueous solution of ethanol.

In order to make the purity of the extracted protein higher, the protein needs to be washed after the protein precipitation and centrifugation, compared with other washing modes, the method of firstly washing the protein with the methyl cellulose aqueous solution and then washing the protein with ethanol can make the purity of the protein reach a higher level, and the methyl cellulose can possibly play a role in maintaining the stability of the protein.

Preferably, the mass fraction of the methylcellulose aqueous solution is 0.5-2%, such as 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2%, or the like.

Preferably, the mass fraction of the ethanol aqueous solution is 75-95%, such as 75%, 78%, 80%, 82%, 85%, 90%, or 95%, etc.

Preferably, the temperature of the washing is 2-6 ℃, such as 2 ℃, 3 ℃, 4 ℃, 5 ℃ or 6 ℃, etc.

As a preferred technical scheme of the invention, the extraction method comprises the following steps:

(1) dehydrating and deoiling fructus Cannabis to make its water content be 5-10% and oil content be 3-5%;

(2) mixing the treated fructus Cannabis with salt solution at a mass volume ratio of 1 (5-15) g/mL, heating and extracting at 60-80 deg.C for 20-40min, and performing ultrasonic extraction at 30-40 deg.C for 60-180min to obtain extractive solution;

(4) And (4) standing and cooling the supernatant obtained in the step (3) to 2-6 ℃, separating out protein, centrifuging, washing with 0.5-2% of methyl cellulose aqueous solution at 2-6 ℃, and washing with 75-95% of ethanol aqueous solution at 2-6 ℃ to obtain the edestin.

In another aspect, the present invention provides a edestin extracted by the above extraction method.

In a further aspect, the invention provides the use of edestin as described above for the preparation of a product for anti-fatigue, immune health improvement and cardiovascular disease prevention.

The globulin extracted by the extraction method has high arginine content, and the arginine can provide nitric oxide, promote vasodilatation, reduce vascular resistance, reduce cardiac output load and relieve the condition of cardiac striation pain; meanwhile, arginine also has an antioxidation effect and can reduce the oxidation of low-density lipoprotein (LDL), so that the probability of small blood vessel obstruction and myocardial necrosis of the heart is reduced. In addition, arginine has functions of improving immune system health and preventing diseases, and can accelerate the healing speed of the body by taking food with high arginine content under the condition of body injury.

Compared with the prior art, the invention has the following beneficial effects:

The extraction method provided by the invention is simple and easy to operate, the yield of the extracted globulin is high, the highest yield can be up to 44.7%, the purity is high, the highest yield can be up to 96%, the globulin contains all essential amino acids of a human body, the arginine content is particularly high, and the globulin can be applied to food, health-care products or medicines and has the effects of resisting fatigue, improving immune health, preventing cardiovascular diseases and the like.

Drawings

FIG. 1 is an infrared spectrum of edestin obtained in example 1;

FIG. 2 is a circular dichroism spectrum of edestin obtained in example 1;

FIG. 3 is an SDS-PAGE gel of edestin prepared in example 3 under reducing conditions;

FIG. 4 is an SDS-PAGE gel of edestin prepared in example 3 under non-reducing conditions.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The protein content determination methods referred to in the following examples all employ Kjeldahl method for determination, and the specific procedures are as follows:

1. reagents and materials

1.1 reagents

Unless otherwise stated, all reagents used in the method are analytically pure, and water is tertiary water specified in GB/T6682.

Copper sulfate (CuSO)₄·5H₂O), potassium sulfate (K)₂SO₄) Sulfuric acid (H)₂SO₄) Boric acid (H)₃BO₃) Methyl red indicator (C)₁₅H₁₅N₃O₂) Bromcresol green indicator (C)₂₁H₁₄Br₄O₅S), methylene blue indicator (C)₁₆H₁₈ClN₃S·3H₂O), sodium hydroxide (NaOH), 95% ethanol (C)₂H₅OH)。

1.2 preparation of reagents

1.2.1 boric acid solution (20 g/L): 20g of boric acid is weighed, dissolved in water and diluted to 1000 mL.

1.2.2 sodium hydroxide solution (400 g/L): 40g of sodium hydroxide was weighed, dissolved in water, cooled and diluted to 100 mL.

1.2.3 sulfuric acid Standard titration solution [ c (1/2H)₂SO₄)]0.0500mol/L or hydrochloric acid standard titration solution [ c (HCl)]0.0500mol/L。

1.2.4 methyl Red ethanol solution (1 g/L): 0.1g of methyl red was weighed, dissolved in 95% ethanol and diluted to 100mL with 95% ethanol.

1.2.5 methylene blue ethanol solution (1 g/L): 0.1g of methylene blue was weighed, dissolved in 95% ethanol and diluted to 100mL with 95% ethanol.

1.2.6 Bromocresol Green ethanol solution (1 g/L): 0.1g of bromocresol green was weighed, dissolved in 95% ethanol and diluted to 100mL with 95% ethanol.

1.2.7A mixing indicator: 2 parts of methyl red ethanol solution are extemporaneously mixed with 1 part of methylene blue ethanol solution.

1.2.8B mixing indicator liquid: 1 part methyl red ethanol solution and 5 parts bromocresol green ethanol solution were mixed extemporaneously.

2. Apparatus and device

2.1 balance: the sensory dose is 1 mg.

2.2 Nitrogen determination distillation apparatus

3. Analytical procedure

3.1.1 sample treatment: 0.2g to 2g (about 30mg to 40mg nitrogen) of a well-mixed solid sample is weighed to an accurate amount of 0.001g, the mixture is transferred into a dry 100mL nitrogen-fixing bottle, 0.4g of copper sulfate, 6g of potassium sulfate and 20mL of sulfuric acid are added, a small funnel is placed at the opening of the bottle after the mixture is lightly shaken, and the bottle is obliquely supported on a foraminous asbestos net at an angle of 45 ℃. Carefully heating, after the contents are completely carbonized and the foam is completely stopped, enhancing the fire power, keeping the liquid in the bottle slightly boiling until the liquid is blue-green, clear and transparent, and continuing to heat for 0.5 h. Taking down and cooling, carefully adding 20mL of water, cooling, transferring into a 100mL volumetric flask, washing the volumetric flask with a small amount of water, merging the washing liquor into the volumetric flask, adding water to the scale, and uniformly mixing for later use. And simultaneously, carrying out a reagent blank test.

3.1.2 determination: installing a nitrogen-fixing distillation device, filling water into the water vapor generator to 2/3, adding a plurality of glass beads, a plurality of drops of methyl red ethanol solution and a plurality of milliliters of sulfuric acid to keep the water acidic, and heating and boiling the water in the water vapor generator to keep boiling.

3.1.3 Add 10.0mL boric acid solution and 2 drops of the A mixing indicator or B mixing indicator into the receiving flask, insert the lower end of the condenser under the liquid surface, accurately suck 2.0mL to 10.0mL sample treatment solution from the cuvette into the reaction chamber according to the nitrogen content in the sample, wash the cuvette with 10mL water and flow it into the reaction chamber, and then plug the rod-shaped glass stopper. 10.0mL of sodium hydroxide solution was poured into a small glass, the plug was lifted to flow slowly into the reaction chamber, immediately capped, and sealed with water. The screw clamp was clamped and distillation commenced. Distilling for 10min, moving the distillate receiving bottle, allowing the liquid surface to leave the lower end of the condenser tube, and distilling for 1 min. Then, the outside of the lower end of the condenser tube was washed with a small amount of water, and the distillate receiver bottle was removed. Titrating with sulfuric acid or hydrochloric acid standard titration solution as soon as possible to the end point, if the indicator solution is mixed with A, the color of the end point is grey blue; when the indicator liquid was mixed with B, the color of the end point was light grayish red. And simultaneously, making a reagent blank.

The protein content of the sample was calculated according to the following formula:

in the formula: x-protein content in the sample in grams per hundred grams (g/100 g);

V₁-the volume of the test solution depleted in sulfuric acid or hydrochloric acid standard titration solution in milliliters (mL);

V₂reagent blank consumption volume of sulfuric acid or hydrochloric acid standard titration solution in milliliters (mL);

c-concentration of sulfuric acid or hydrochloric acid standard titration solution in moles per liter (mol/L);

0.014-1.0 mL sulfuric acid [ c (12H)₂SO₄)＝1.000mol/L]Or hydrochloric acid [ c (HCl) ═ 1.000mol/L]Mass of nitrogen equivalent to standard titration solution in grams (g);

m-mass of the sample in grams (g);

V₃-aspirating the volume of digestive juices in milliliters (mL);

f-coefficient of conversion of nitrogen to protein;

100-conversion factor.

When the protein content is more than or equal to 1g/100g, three effective figures are reserved; at protein content <1g/100g, two significant digits are retained.

Note: when only the nitrogen content is detected, the protein conversion factor F need not be multiplied.

Example 1

This example provides a method for extracting edestin, which comprises the following steps:

(1) dehydrating and deoiling fructus Cannabis to make its water content be 8% and oil content be 4%;

(2) mixing 100g of the treated fructus cannabis with a salt solution according to a mass-volume ratio of 1:10g/mL, firstly heating and extracting at 70 ℃ for 30min, and then performing ultrasonic extraction at 35 ℃ for 120min, wherein the ultrasonic power is 800W/L to obtain an extracting solution; the salt solution is a salt solution system formed by mixing 5% of sodium chloride, 2% of ammonium sulfate, 0.5% of hyaluronic acid and 0.5% of trehalose.

(4) and (3) standing and cooling the supernatant obtained in the step (3) to 4 ℃, separating out protein, centrifuging, washing with 1% methyl cellulose aqueous solution at 4 ℃, then washing with 90% ethanol aqueous solution at 4 ℃ to obtain 41.6g of the hemp seed globulin, and detecting the protein purity to be 96% by using a Kjeldahl method.

Example 2

(1) dehydrating and deoiling fructus Cannabis to make its water content be 5% and oil content be 5%;

(2) mixing 100g of the treated fructus cannabis with a salt solution according to a mass-volume ratio of 1:5g/mL, firstly heating and extracting at 60 ℃ for 40min, and then performing ultrasonic extraction at 40 ℃ for 60min, wherein the ultrasonic power is 800W/L to obtain an extracting solution; the salt solution is a salt solution system formed by mixing 6% of sodium chloride, 1% of ammonium sulfate and 1% of hyaluronic acid.

(4) and (3) standing and cooling the supernatant obtained in the step (3) to 4 ℃, separating out protein, centrifuging, washing with 0.5% methyl cellulose aqueous solution at 4 ℃, then washing with 75% ethanol aqueous solution at 4 ℃ to obtain 43.7g of the hemp seed globulin, and detecting the protein purity to be 95% by using a Kjeldahl method.

Example 3

(1) dehydrating and deoiling fructus Cannabis to make its water content be 10% and oil content be 3%;

(2) mixing 100g of the treated fructus cannabis with a salt solution according to a mass-volume ratio of 1:15g/mL, firstly heating and extracting at 80 ℃ for 20min, then performing ultrasonic extraction at 30 ℃ for 180min, wherein the ultrasonic power is 800W/L, and obtaining an extracting solution; the salt solution is a salt solution system formed by mixing 3% of sodium chloride, 4% of ammonium sulfate and 1% of trehalose.

(4) and (3) standing and cooling the supernatant obtained in the step (3) to 4 ℃, separating out protein, centrifuging, washing with 2% methyl cellulose aqueous solution at 4 ℃, then washing with 95% ethanol aqueous solution at 4 ℃ to obtain 44.7g of the hemp seed globulin, and detecting the protein purity to be 93% by using a Kjeldahl method.

Example 4

This example provides a method for extracting edestin, which is different from example 1 only in that the edestin is dehydrated and deoiled in step (1) to make the water content of the edestin 8% and the oil content of the edestin 8%, and other conditions are kept the same. 35.8g of the hemp seed globulin is obtained, and the protein purity is 92% by using a Kjeldahl method.

Example 5

This example provides a method for extracting edestin, which is different from example 1 only in that the edestin is dehydrated and deoiled in the step (1) to make the water content of the edestin 12% and the oil content of the edestin 4%, and other conditions are kept consistent. 28.9g of the edestin is obtained, and the protein purity is 88 percent by using a Kjeldahl method.

Example 6

This example provides a method for extracting edestin, which is different from example 1 only in that 100g of the processed fructus cannabis is mixed with a salt solution in a mass-to-volume ratio of 1:5g/mL in step (2), and other conditions are kept consistent. 36.4g of the edestin is obtained, and the protein purity is 93 percent by using a Kjeldahl method.

Example 7

This example provides a method for extracting edestin, which is different from example 1 only in that a single heating extraction method is used for extraction in step (2), and the extraction is performed at 70 ℃ for 150min, and other conditions are consistent. 27.8g of the edestin is obtained, and the protein purity is 93 percent by using a Kjeldahl method.

Example 8

This example provides a method for extracting edestin, which is different from example 1 only in that a single ultrasonic extraction mode is adopted in step (2), ultrasonic extraction is performed at 35 ℃ for 150min, and other conditions are consistent. 33.5g of the hemp seed globulin is obtained, and the protein purity is 90 percent by using a Kjeldahl method.

Example 9

This example provides a method for extracting edestin, which only differs from example 1 in that a two-stage extraction is still used in step (2), but the temperature for heating extraction is 90 ℃ and the time is 15min, and other conditions are kept consistent. 42.6g of the hemp seed globulin is obtained, and the protein purity is 90 percent by using a Kjeldahl method.

Example 10

This example provides a method for extracting edestin, which only differs from example 1 in that a two-stage extraction is still used in step (2), but the temperature for heating extraction is 50 ℃ and the time is 50min, and other conditions are kept consistent. 39.6g of the edestin is obtained, and the protein purity is 94% by Kjeldahl method.

Example 11

This example provides a method for extracting edestin, which only differs from example 1 in that a two-stage extraction method is still used in step (2), but the temperature of ultrasonic extraction is 50 ℃, the time is 50min, and other conditions are consistent. 41.2g of the edestin is obtained, and the protein purity is 92% by using a Kjeldahl method.

Example 12

This example provides a method for extracting edestin, which only differs from example 1 in that a two-stage extraction method is still used in step (2), but the temperature of ultrasonic extraction is 20 ℃ and the time is 200min, and other conditions are kept consistent. 39.8g of the edestin is obtained, and the protein purity is 93 percent by using a Kjeldahl method.

Example 13

This example provides a method for extracting edestin, which is different from example 1 only in that the salt solution in step (2) is a salt solution system formed by mixing 5% of sodium chloride and 2% of ammonium sulfate, and other conditions are consistent. 42.5g of the hemp seed globulin is obtained, and the protein purity is 92 percent by using a Kjeldahl method.

Example 14

This example provides a method for extracting edestin, which is different from example 1 only in that the salt solution in step (2) is a salt solution system formed by mixing 8% sodium chloride and 5% ammonium sulfate, and other conditions are consistent. 44.4g of the hemp seed globulin is obtained, and the protein purity is 90 percent by using a Kjeldahl method.

Example 15

This example provides a method for extracting edestin, which differs from example 1 only in that the salt solution in step (2) is a 7% sodium chloride salt solution system, and other conditions are kept the same. 38.2g of the edestin is obtained, and the protein purity is 91% by using a Kjeldahl method.

Example 16

This example provides a method for extracting edestin, which is different from example 1 only in that the salt solution in step (2) is a 7% ammonium sulfate salt solution system, and other conditions are consistent. 41.5g of the hemp seed globulin is obtained, and the protein purity is 92% by using a Kjeldahl method.

Example 17

This example provides a method for extracting edestin, which differs from example 1 only in that in step (4) it is washed with water at 4 ℃ and then with 90% aqueous ethanol at 4 ℃, and the other conditions are kept the same. 42.8g of the hemp seed globulin is obtained, and the protein purity is 92% by using a Kjeldahl method.

The yields and purities of edestin extracted in examples 1-17 are summarized in Table 1.

TABLE 1

As can be seen from the data in Table 1: the extraction method has high yield, and the extracted edestin has high purity, so the extraction method is an ideal extraction method of the edestin. As can be seen from the comparison of example 1 and examples 4-5, the water content and oil content of the hemp seed raw material have an influence on the yield and purity of the product; comparing example 1 with example 6, it can be known that the feed-liquid ratio in the extraction process is also one of the key factors influencing the yield and purity of the protein; comparing example 1 with examples 7-8, it can be seen that the two-stage extraction method adopted in the present invention is significantly superior to the single heating extraction method or the single ultrasonic extraction method; in comparison with example 1 and examples 9 to 12, the temperature during the extraction process is also one of the key factors affecting the yield or purity of the product; as can be seen from comparison of examples 1 and 13, the protein purity is significantly improved by adding a small amount of hyaluronic acid and/or trehalose to the conventional salt solution extraction system; the data of example 15 and example 16 show that the protein yield of the single salt solution system is lower than that of the mixed salt system of the present invention; comparing example 1 with example 17, it can be seen that the protein purity is also affected by the washing process, and the protein purity can be maintained at a high level by adding a small amount of methylcellulose to the conventional aqueous solution.

Example 18

In this example, infrared spectroscopy of the edestin prepared in example 1 was carried out, and as shown in fig. 1, it can be seen that: 1660cm^-1The position is an amide I band, and the stretching vibration peak of carbonyl; 1531cm^-1In the form of an amide II band, NH₂The in-plane deformation vibration peak of (1); 1252cm^-1The position is an amide III band, and the absorption peak of C-N is shown. The above are typical absorption peaks of proteins.

Example 19

In this example, the edestin prepared in example 1 was subjected to circular dichroism spectroscopy, and as shown in FIG. 2, the percentages of α -helix, β -sheet, β -turn and random coil were about 8.1%, 28.8%, 25.9% and 37.7%, respectively. The prepared fructus cannabis globulin is proved to have compact structure.

Example 20

In this embodiment, the hemp seed globulin prepared in example 2 is analyzed for polysaccharide content, moisture content, dietary fiber content and amino acid content, and the specific operation method is as follows:

the detection of the water content meets the national standard: GB 5009.3-2016;

the detection of the content of the dietary fiber meets the national standard: GB 5009.88-2014;

the detection of the amino acid content meets the national standard GB 5009.124-2016;

and (3) detecting the content of polysaccharide: the phenol-sulfuric acid spectrophotometry method specifically comprises the following steps:

weighing appropriate amount (m) of fructus Cannabis protein powder ₁) Put into a 100mL volumetric flask, add 80mL of water, heat in a boiling water bath for 1 hour, add pure water to make up 100 mL. Centrifuging and taking a proper amount of supernatant (V)₁) Placing in a 50mL centrifuge tube, adding 20mL absolute ethyl alcohol, standing in a refrigerator at 4 ℃ for 4h, centrifuging to leave precipitate, washing with 80% ethanol for three times, dissolving with water to constant volume (V)₂)。

Taking a proper amount of supernatant (V)₃) Placing in a 25mL colorimetric tube, adding pure water to a constant volume of 1mL, adding 1mL of 6% phenol solution, adding 5mL of concentrated sulfuric acid, boiling in a water bath for 20min, and measuring the light absorption value at 490 nm.

The results showed that the content of polysaccharide in the edestin was 1.6%, the content of water was 2.8%, and the content of insoluble cellulose was 4.5%, and the composition ratio of amino acids in 1.2mg of edestin was as shown in table 2, in which the content of arginine was 15% or more.

TABLE 2

Example 21

In this example, the edestin prepared in example 3 was subjected to SDS-PAGE to detect the protein.

(1) Glue making

The reagent adopts a Solibao gel preparation kit (P1200) to prepare separation gel, 30 percent of separation gel, 1.5M tris-HCL, 10 percent SDS and dd water are sequentially added, after being fully and uniformly mixed, a PAGE gel coagulant and a PAGE gel coagulant are added, and the mixture is uniformly mixed (the gel coagulant and the gel coagulant are ensured to be added finally). Slowly move into the interlayer of the glass plate. Then slowly adding dd water to the overflow surface, standing for 30-60min, and ensuring the surface of the separation gel to be flat. And (3) preparing a concentrated gel, pouring out water, sequentially adding 30% of separation gel, 1M tris-HCL, 10% SDS and dd water, fully and uniformly mixing, then adding a PAGE gel coagulant and a PAGE gel coagulant, and uniformly mixing (ensuring that the gel coagulant and the PAGE gel coagulant are added finally). Slowly move into the interlayer. Inserting a glue making comb.

(2) Sample preparation

The reagent comprises 5X protein loading buffer solution (containing DTT), 4X protein loading buffer solution, protein solution 80-20 μ L loading buffer solution (containing DTT), and boiling with boiling water for 3-5 min; or protein solution 60-20 μ L loading buffer solution, boiling with boiled water for 3 min. Sample application is carried out by 10 mu L of sample, and sample application is fast by 6-10 mu L of marker.

(3) Electrophoresis

The initial voltage was 30v, the blue horizontal line reached the level of the separation gel, 100v was used instead, and the electrophoresis was terminated when the blue horizontal line reached the bottom. Electrode buffer (5 ×): 125mM Tris, 1.25M glycine, 0.5% (W/V) SDS. When in use, 100mL of the solution is taken and the volume is up to 500 mL.

(4) Dyeing and decolouring

Dyeing with Coomassie brilliant blue dye solution for half an hour, boiling with boiled water for decolorizing, and changing the liquid once in 10min until decolorizing is clear.

The electrophoretogram of edestin under reducing conditions is shown in FIG. 3. As can be seen from fig. 3: in the gel electrophoresis image band in the reduction state, the molecular weight is about 35k, and the molecular weight is the acid group molecular weight of globulin; 19k, 18k are the molecular weight of the globin bases, DTT opens a disulfide bond, and one subunit of the protein breaks down into a 35k acid group and a 19k or 18k base.

The electrophoretogram of edestin under non-reducing conditions is shown in FIG. 4. As can be seen from fig. 4: in the band of the electrophoretogram under the non-reducing condition, the molecular weight of about 50k is the molecular weight of one subunit of the protein.

The applicant states that the present invention is illustrated by the above examples to describe the extraction method of edestin, the product and the application of the present invention, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must rely on the above examples to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. A method for extracting edestin is characterized by comprising the steps of dehydrating and deoiling fructus cannabis, mixing with a salt solution, extracting, filtering and cooling to obtain the edestin.

2. The extraction method according to claim 1, characterized in that it comprises the steps of:

(1) dehydrating and deoiling fructus cannabis;

3. The extraction method according to claim 1 or 2, wherein the deoiling manner in the step (1) comprises any one or a combination of at least two of a mechanical method, a solvent extraction method or a supercritical extraction method;

preferably, the mechanical process comprises a mechanical cold-pressing process;

preferably, the solvent extraction method comprises n-hexane and/or petroleum ether;

preferably, the moisture content of the dehydrated and deoiled fructus cannabis in the step (1) is 5-10%, and the oil content is 3-5%.

4. The extraction method according to any one of claims 1 to 3, wherein the solvent of the salt solution in the step (2) is water, and the solute comprises any one or a combination of at least two of sodium chloride, ammonium sulfate, magnesium chloride, calcium chloride or sodium sulfate;

Preferably, the solvent of the salt solution in the step (2) is water, and the solute comprises sodium chloride and ammonium sulfate;

preferably, the mass concentration of the sodium chloride in the salt solution is 3-6%, and the mass concentration of the ammonium sulfate in the salt solution is 1-4%;

preferably, the salt solution in the step (2) also contains hyaluronic acid and/or trehalose;

preferably, the mass fraction of the hyaluronic acid in the salt solution is 0.1-1%;

preferably, the mass fraction of the trehalose in the salt solution is 0.1-1%.

5. The extraction method according to any one of claims 1 to 4, wherein the mass-to-volume ratio of the hemp seed after the treatment in the step (2) to the salt solution is 1 (5-15) g/mL.

6. The extraction method according to any one of claims 1 to 5, wherein the extraction of step (2) comprises a two-stage extraction process, the first stage extraction being a heating extraction and the second stage extraction being an ultrasonic extraction;

preferably, the temperature of the heating extraction is 60-80 ℃;

preferably, the time for heating and extracting is 20-40 min;

preferably, the temperature of the ultrasonic extraction is 30-40 ℃;

preferably, the time of ultrasonic extraction is 60-180 min;

Preferably, the power of the ultrasound is 300-1000W/L, preferably 800W/L.

7. The extraction method according to any one of claims 1 to 6, wherein the cooling in the step (4) is cooling to 2 to 6 ℃;

preferably, the protein is precipitated in step (4) and then centrifuged and washed;

preferably, the washing comprises washing with a methyl cellulose aqueous solution and then washing with an ethanol aqueous solution;

preferably, the mass fraction of the methyl cellulose aqueous solution is 0.5-2%;

preferably, the mass fraction of the ethanol water solution is 75-95%;

preferably, the temperature of the washing is 2-6 ℃.

8. The extraction method according to any one of claims 1 to 7, characterized in that it comprises the steps of:

(2) mixing the treated fructus Cannabis with salt solution at a mass volume ratio of 1 (5-15) g/mL, extracting at 60-80 deg.C for 20-40min, and extracting at 30-40 deg.C for 60-180min to obtain extractive solution;

9. Edestin extracted by the extraction method according to any one of claims 1-8.

10. Use of edestin according to claim 9 for the preparation of a product for anti-fatigue, immune-health improvement, cardiovascular disease prevention.